Phase variable colony morphotypes of Clostridioides difficile elicit distinct host responses during acute infection

Many Clostridioides difficile strains can form two colony morphotypes: rough and smooth. The rough and smooth morphotypes differ in multiple phenotypes, including cell length and chaining, motility, biofilm production, and virulence in the hamster model of C. difficile infection (CDI). Colony morphology undergoes phase variation and is determined by the ON/OFF expression of cmrRST, which encodes a signal transduction system. Here, we test the hypothesis that differences in colony morphology and the associated phenotypes influence pathogenesis and the host response to infection. We first compared the rough and smooth colony variants of wild-type C. difficile in a mouse model of CDI. However, CmrRST phase varied during infection such that the C. difficile populations became indistinguishable in feces and tissues, and no differences in disease were observed in mice inoculated with these variants. We next circumvented phase variation using mutants that form only rough or only smooth colonies. Co-infection of mice with these phenotypically locked strains revealed that the smooth colony mutant has greater fitness than the rough mutant, which is outcompeted during late infection. In addition, NanoString analyses showed a higher number of differentially expressed pro-inflammatory genes and overall higher expression levels in mice infected with the rough colony mutant, independent of bacterial burden and toxin levels. Our results indicate that in a mouse model of CDI, cells from rough colonies are more immunostimulatory during early murine infection, potentially leading to reduced relative fitness compared to cells from smooth colonies. IMPORTANCEClostridioides difficile is a major cause of antibiotic-associated diarrhea and one of the most common hospital-acquired infections. These infections are often recurrent and recalcitrant to antibiotic treatment. Bacteria use diverse mechanisms to adapt to stressful host environments, including the development of different subpopulations to help ensure survival of the whole population. C. difficile produces two different colony types, rough and smooth, that have multiple distinguishing traits. We interrogated the effect of these different forms of C. difficile on pathogenesis and the host immune response. We show that the rough colony phenotype results in a more robust immune response and has reduced fitness during infection compared to the smooth colony phenotype. This work sheds light on the impact of these phenotypic subpopulations on disease, leading to a deeper understanding of C. difficile-host interactions.